Detecting species-specific bacterial meningitis markers with iDDS probes Confidential PI: Shafer, David A., PhD PROJECT SUMMARY Rapid and accurate molecular diagnostics assays empower physicians to make informed treatment decisions. Meningitis may be caused by infection with microorganisms such as viruses, gram-negative (GN) and gram-positive (GP) bacteria, fungi, or parasites. Most frequently, meningitis results from infections with non-polio enteroviruses (EV) and parechoviruses (PV), which typically self-resolve within 10 days. In contrast, bacterial meningitis has a high mortality rate that approaches 100% if not treated. Meningitis patients present with similar symptoms regardless of origin; thus, timely diagnosis of the causative agent is paramount for patient care. Patients are commonly treated with an ?empiric? regimen of antibiotics until a definitive diagnosis can be made. Nucleic acid amplification tests (NAATs) reduce diagnosis turnaround times by several days compared to standard culturing methods, allowing shortened hospital stays, appropriate use of antibiotics, and a reduced financial burden to patients and the health care system. Currently, there are only two FDA-approved NAATs for diagnosing viral and/or bacterial meningitis. Unfortunately, these tests are only approved for use on the manufacturer?s fully automated systems (the costs of which are often prohibitive) and may not determine the etiological agent of bacterial meningitis. GeneTAG Technology, Inc. has developed instrument- independent, error-preventing DNA Detection Switch (iDDS) probes, which employ a fluorescent labeled probe and a slightly mismatched quencher-labeled antiprobe. In the absence of the intended target, the antiprobes hybridize to the probes, quenching their fluorescence and preventing off-target detection. Recently, we developed EV and PV assays employing two iDDS probes for each target, providing simultaneous 2-color signaling and automatic confirmation of positive or negative test results. We have also developed iDDS probes for GN and GP detection. In the proposed studies, we are interested in developing a reflex test that can be used to determine which of 7 common bacterial species is present, based on the detection of species-specific genetic markers for each bacterial species. Such a reflex test is important because knowing the bacterial species can guide physicians in prescribing appropriate antibiotic treatment. After initial validation studies, this test will be re-formulated into a lyophilized format where all reagents, primers, and probes are included, and the test would only require adding sample and molecular-grade water. The lyophilized test will be re-evaluated with genomic DNA samples representing each bacterial test species and cerebrospinal fluid specimens. Ultimately, we are interested in gaining FDA approval for our lyophilized meningitis assay. Delivering a high fidelity, cost-effective meningitis assay should positively impact meningitis diagnostics, guide physicians in selecting appropriate therapy, and provide a direct benefit to public health.